Project Summary Our overall goal is to understand the structural mechanisms for the functional dysregulation of the cardiac ryanodine receptor (RyR2) Ca2+-release channels in the sarcoplasmic reticulum (SR). RyR2 contributes to and is a therapeutic target for treating heart failure (HF) and arrhythmias. The proposed studies take the new knowledge obtained in the current award period to new levels of insight with novel methods and goals (below). Our work has developed a new integrated mechanistic paradigm explaining how RyR2 becomes dysfunctional and promotes arrhythmias, and systolic and diastolic dysfunction in HF. We discovered a common pathological RyR2 structural state that causes diastolic SR Ca leak, identified key physiological modulators of this state ? calmodulin (CaM), ROS, Ca/CaM-dependent kinase (CaMKII) ?, and devised novel drug discovery strategies to target leaky RyR2 conformation. The core observation: CaM inhibits (a) RyR2 Ca leak and (b) RyR2 binding of DPc10 biosensor peptide. In HF, RyR2 readily binds DPc10 but CaM affinity is reduced and Ca leak is high. These 3 HF RyR2 properties can be induced by CaMKII and ROS (both increased in HF) and DPc10, but dantrolene or high [CaM] reverses each of them. Combining this conformation-shifting paradigm with our FRET-based molecular tools has enabled us to start a drug discovery program to seek novel therapeutics for HF and CPVT, and this is already yielding promising compounds. The influence of CaM-CaMKII-RyR2 interactions on cardiac function and dysfunction are widely appre- ciated. Here, we extend our mechanistic progress to determine the structural basis of functional CaM-CaMKII- RyR2 effects. We develop a new FRET-based dynamic conformational approach with functioning RyRs, to test hypotheses suggested by cryo-EM static structural snapshots of frozen, detergent-purified RyR. For example, cryo-EM and FRET-based mapping place apo(Ca-free)-CaM in similar locations on RyR1 (skeletal muscle) and RyR2 (heart), but the RyR2 position of Ca-CaM is controversial. We found that human CaM mutants linked to CPVT outcompete WT-CaM/RyR2 binding, but promote (rather than inhibit) arrhythmogenic SR Ca leak. This apo-CaM effect is akin to RyR1 activation by WT-CaM. So structure-function analysis of WT and CPVT- CaMs (as we plan) may be the key to expose the molecular mechanism of CaM-RyR regulation. We will test this using our evolving RyR-targeted FRET toolkit to correlate state-of-the-art confocal studies in relatively intact cardiomyocytes (Bers Lab) with fluorescence lifetime detection of FRET within RyR2 in SR vesicles (Cornea Lab) and emerging high-resolution structural information. Our success with RyR-CaM-FKBP, the importance of CaMKII in RyR regulation, and our parallel CaMKII FRET tool development, drive us to enhance understanding of dynamic CaMKII with new mechanistic clarity. Specific Aims are to resolve the (1) structural basis of CaM regulation of RyR2 vs. RyR1, (2) Ca-dependent kinetics of CaM-RyR2 structure-function in SR and myocytes, (3) differences in CaMKII physical states upon direct CaM and autonomous activation.